KR101209835B1 - Quantification of 17α-hydroxyprogesterone in serum by isotope dilution-matrix assisted laser desorption ionization-mass spectrometry - Google Patents

Abstract

The present invention relates to a rapid and accurate method of quantifying 17α-hydroxyprogesterone in serum.
The present invention provides a method for effectively quantitatively analyzing 17α-hydroxyprogesterone, which is a representative subsignal hormone changed from serum by congenital adrenal hyperplasia, without developing a quantitative curve, thereby developing congenital adrenal hyperplasia. It can be evaluated quickly and accurately.

Description

Quantification of 17α-hydroxyprogesterone in serum by isotope dilution-matrix-based laser desorption / ionization mass spectrometry {quantification of 17α-hydroxyprogesterone in serum by isotope dilution-matrix assisted laser desorption ionization-mass spectrometry}

The present invention relates to a rapid and accurate method of quantifying 17α-hydroxyprogesterone in serum.

Recently, in addition to the research areas of genomics and proteomics, researches for diagnosing and preventing diseases have been widely conducted through measuring metabolic techniques that can comprehensively understand bioactive compounds in vivo. When measuring the concentration of steroid hormones present in small amounts of plasma, liquid / gas chromatography, which has excellent sensitivity and separation, is often used, but it takes time to separate the analyte from the interferences present in complex biological samples. This can lead to lower analyte stability [Rapid Commun Mass Spectrom., 2009, 23: 1783-1791; Anal Chem., 2007, 79: 6020-6026. Therefore, matrix-based laser desorption / ionization mass spectrometry can be applied to analyze analytes in a short time after sample preparation [Anal Chem., 2006, 78: 164-173]. However, this has a problem in that the reproducibility is lowered compared to the existing methods in the quantitative analysis, there is a problem in its application.

Meanwhile, in the case of congenital adrenal hyperplasia (CAH), a disorder occurs in the process of cortisol formation in the adrenal cortex, which is a function of the steroid 21-hydroxylase, a cytochrome P450 enzyme. The incidence rate by drop is 95% or more. A deficiency of 21-hydroxylase decreases the production of cortisol and aldosterone in the adrenal glands, and at the same time, the adrenal cortex is over-produced due to hypersecretion of the adrenal cortical stimulating hormone (ACTH), which promotes the production of androgenic hormones in the adrenal glands. [Endo. Rev., 2000, 21: 245-279. To date, the diagnosis of 21-hydroxylase deficiency has shown that the concentration of 17α-hydroxyprogesterone (17α-hydroxyprogesterone, 17α-OHP) produced at the precursor stage of 21-hydroxylation as shown in FIG. It is measured by.

In general, if the concentration of 17α-hydroxyprogesterone in the serum is more than 20 ~ 100 ng / mL, it can be diagnosed as congenital adrenal hyperplasia, and the test method is radioimmunoassays (RIA), fluoroimmunoassays (FIA) ), Enzyme-linked immunosorbent assay (EIA), and the like. Enzyme-immunity affinity methods for measuring the activity of such enzymes have the disadvantage of low reproducibility of experimental results under various conditions such as various experimental conditions [Cancer Epidemiol. Biomarkers Prev., 2007, 16: 1004-1008; Ann. Clin. Biochem., 2008, 45: 380-388. In addition, these test methods have been changed in many cases, such as birth weight, gestational age, premature infants, infection, respirator treatment, etc., and the need for retesting is increasing [Rev. Endocr. Metab. Disord., 2007, 8: 349-363]. Therefore, there is a need for a quantitative analysis technique for rapidly and accurately measuring 17α-hydroxyprogesterone in serum.

Therefore, the present inventors have conducted research and efforts to measure 17α-hydroxyprogesterone in serum more quickly and accurately, and as a result, a solid phase extraction process from serum added with isotopically substituted 17α-hydroxyprogesterone- d ₈ as an internal standard, After re-extraction through liquid-liquid extraction, the present invention was completed by discovering that the selective detection of the extract using a laser desorption / ionization mass spectrometer allows quantitative analysis of 17α-hydroxyprogesterone.

Accordingly, an object of the present invention is to provide a method for rapidly and accurately quantitatively evaluating 17α-hydroxyprogesterone in serum.

The present invention,

Adding 17α-hydroxyprogesterone- d ₈ with isotopic substitution to the serum as an internal standard;

Extracting the hormone from the serum with a lipophilic and hydrophilic copolymer adsorbent and then re-extracting with an organic solvent; And

Crystallization by mixing the re-extracted analyte and α-cyano-4-hydroxy cinnamic acid, and selectively detecting the hormone using a laser desorption / ionization mass spectrometer step

Characterized in that the method of quantitative analysis of 17α-hydroxyprogesterone in serum comprising a.

The present invention provides a method for effectively quantitatively analyzing 17α-hydroxyprogesterone, which is a representative subsignal hormone changed from serum by congenital adrenal hyperplasia, without developing a quantitative curve, thereby developing congenital adrenal hyperplasia. It can be evaluated quickly and accurately.

1 is a schematic diagram showing the process of metabolism of 17α-hydroxyprogesterone in vivo by 21-hydroxylase, the main cause of congenital adrenal hyperplasia.
2 is a mass spectrometry spectrum of an analyte and an isotope-substituted internal standard identified through the matrix-based laser desorption / ionization mass spectrometry of the examples, and m / z 253.15 and 277.22 are 17α-hydroxyprogesterone and 17α-hydr, respectively. medroxyprogesterone - shows the amount of the ion-d _8.

The present invention will be described in detail as follows.

According to the present invention, hormones are extracted from serum added with isotope-substituted 17α-hydroxyprogesterone- d ₈ as an internal standard through solid phase extraction and liquid-liquid extraction, and then detected by laser desorption / ionization mass spectrometry. A method for quantitatively analyzing 17α-hydroxyprogesterone.

First, 17α-hydroxyprogesterone- d ₈ having an isotope substituted in the serum to be analyzed is added as an internal standard.

In this case, the amount of serum used may be 0.1 ~ 2.0 mL, but 0.2 ~ 1.0 mL is preferable considering the problems of analysis sensitivity and sampling.

In addition, the internal standard is preferably added at a concentration of 20 to 200 ng / mL in serum, considering that the average concentration of patients diagnosed with congenital adrenal hyperplasia is about 100 ng / mL. More preferably, it is added at a concentration of / mL.

The serum to which the internal standard is added is preferably put in a buffer solution of pH 4-7 and stored at room temperature for 1-60 minutes to release the protein. The step of releasing the protein releases the analyte that is non-selectively bound to the protein to allow more accurate quantitative analysis in serum.

Thereafter, the hormone is extracted from the serum by a solid phase extraction method using a copolymer adsorbent having lipophilic properties and hydrophilicity, and then extracted again using a liquid-liquid extraction method at an pH of 8 to 12 with an organic solvent. At this time, the organic solvent that can be used is not limited in kind, but preferably one or two selected from methyl tert -butyl ether (MTBE), ethyl acetate, and n-hexane It is preferable to use mixtures of species or more.

The re-extract is evaporated and dried, and the analyte and the α-cyano-4-hydroxy cinnamic acid (α-cyano-4-hydroxy cinnamic acid), which is mainly used in the analysis of fatty compounds, is mixed and at room temperature for 1 to 20 minutes. It is left to crystallize. The crystallization is preferably performed by centrifuging the mixed solution of the analyte and the α-cyano-4-hydroxy cinnamic acid and drying at room temperature.

Thereafter, 17α-hydroxyprogesterone is selectively detected using a laser desorption / ionization mass spectrometer.

Through this process, 17α-hydroxyprogesterone can be quickly quantitated by directly comparing the analytical sensitivity of the analyte and the internal standard without using a separate quantitative calibration curve.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited to the following examples.

Example: Quantitative Analysis of 17α-hydroxyprogesterone

1) Addition of Internal Standard

All steroid hormone-free serums (Scipac, Sittingbourne, UK), including 17α-hydroxyprogesterone, were purchased, and 17α-hydroxyprogesterone (17α-OHP, 2 μg / mL) 20 μL of isotope-substituted internal standard (17α-OHP- d ₈ , 2 μg / mL) was added to prepare the concentration of 17α-OHP and 17α-OHP- d ₈ in serum to 100 ng / mL. It was.

2) Vitrification of Proteins in Serum Samples

To 0.4 mL of serum sample to which isotopically substituted internal standards were added, 2.6 mL of 0.2 M sodium acetate buffer was added to adjust the pH to 5.7 to 6.0. Then, the protein was stored at room temperature for 10 minutes to release the protein.

3) Solid phase extraction

The solid phase extraction method uses an Oasis HLB cartridge [Oasis HLB ^™ , 60 mg, Waters, Co., Milford, MA, USA]. Methanol and 2 mL of distilled water were flowed into the cartridge, respectively, and the serum sample passed through 2) was flowed into the cartridge. To remove impurities, 2 mL of distilled water was poured, followed by 4 mL of methanol to elute the decomposition product of 2) adsorbed to the cartridge, and the eluate was received in a clean test tube. The methanol eluate received in the test tube was evaporated at 40 ° C. through a rotary evaporator.

4) Liquid-Liquid Extraction

1 mL of 0.2 M sodium acetate buffer was added to the test tube of evaporated 3), 2.5 mL of a mixed solvent of ethyl acetate and normal hexane (2: 3, v / v) were added thereto, and stirred for 10 minutes. An organic solvent layer was aliquoted. The fractionated solution was evaporated using nitrogen gas at 40 ° C., which was then dried sufficiently in a vacuum desiccator for 30 minutes or more using P ₂ O ₅ / KOH.

5) Matrix-based laser desorption / ionization mass spectrometry

To analyze 17α-OHP present in serum using matrix-based laser desorption / ionization mass spectrometry, an α-cyano-4-hydroxy cinnamic acid (CHCA) matrix was prepared.

The CHCA matrix was prepared by adding 70% acetonitrile containing 0.2% trifluoroacetic acid so that the concentration of CHCA was 10 mg / mL. 10 μL of the prepared CHCA matrix was mixed with 10 μL of the sample prepared in 4) to obtain an analytical sample.

1.5 mL centrifuge tubes (Durapore PVDF; Millipore, Billerica, MA, Equipped with a polyvinylidene fluoride membrane filter (0.1 μm)) to form uniform crystals in crystallization of the analyte. USA) was added to the analytical sample, centrifuged at 1000 rpm for 30 seconds, and then 0.5 μL of the centrifuged analytical sample was injected into the sample injection plate and dried at room temperature for 10 minutes or more.

Pulsed UV / MALDI using Thermo MALDI (Thermo Fisher, San Jose, CA, USA) connected with Linear Ion Trap Quadrupole Mass Analyzer for the analysis of 17α-OHP and internal standard 17α-OHP- d ₈ (Nitrogen) was analyzed under the conditions shown in Table 1 below with a laser.

Ionization conditions Positive ionization Ion selection condition Selected Reaction Monitoring (SRM) Fixed ion 17α-OHP ( m / z 253)
17α-OHP- d ₈ ( m / z 277) Ionization Collision Energy 17α-OHP (70%)
17α-OHP- d ₈ (75%) Mass scan range m / z 200 ～ 400

6) Quantitative analysis of 17α-hydroxyprogesterone

The mass spectrometry obtained by the above procedure is shown in FIG. 2. In the mass spectrometry, the concentration of analyte 17α-OHP is an internal standard by comparing the intensity of the quantitative ions of each of the 17α-OHP and 17α-OHP- d ₈ compounds which show a difference in mass values, It was confirmed that it can be measured quickly from the concentration of.

Experimental Example 1

Using the analytical method of the above example, analyte 17α-OHP and 17α-OHP- d ₈ were added to 20, 30, 50, 100 and 200 ng / mL, respectively, to the steroid-free serum.

To determine the reproducibility of the 17α-OHP concentration measurement, the detection rate of the analyte and the internal standard was measured, and the reproducibility experiment was performed three times on the same intra-day and three different inter-day. The results are shown in Table 2.

As shown in Table 2, 17α-OHP and 17α-OHP- d ₈ were added at five different concentrations so that the molar ratio was 1, and the results were quantitatively analyzed. The results were shown, in particular, it was confirmed that the most reproducible results were obtained when 17α-OHP- d ₈ was added at a concentration of 100 ng / mL.

Experimental Example  2

In the serum obtained from 10 patients suspected of congenital adrenal hyperplasia (CAH) by the enzyme-linked immunosorbent adsorption method, the 17α-OHP concentration was measured through the procedure of the above example using the analytical method of the above example. 17α-hydroxyprogesterone at a concentration of 112.3 ng / mL was measured from the patient, corresponding to the 17α-OHP concentration range of commonly known patients with congenital adrenal hyperplasia.

Claims (4)

Adding 17α-hydroxyprogesterone- d ₈ with isotopic substitution to the serum as an internal standard;
Extracting 17α-hydroxyprogesterone and 17α-hydroxyprogesterone- d ₈ from the serum with a lipophilic copolymer adsorbent and re-extracting with an organic solvent; And
Crystallized by mixing the re-extracted analyte and α-cyano-4-hydroxy cinnamic acid (alpha) -cyano-4-hydroxy cinnamic acid, using a laser desorption / ionization mass spectrometer and 17α-hydroxyprogesterone Quantitative step of detecting 17α-hydroxyprogesterone- d ₈ ;
Method for quantitative analysis of 17α-hydroxyprogesterone in serum comprising a.
The method according to claim 1, wherein the serum to which the internal standard is added in a buffer solution of pH 4-7 and stored for 1 to 60 minutes at room temperature to release the protein quantitative analysis of 17α-hydroxyprogesterone in serum Way.
The method of claim 1, wherein the crystallization is a quantitative analysis method of 17α-hydroxyprogesterone, characterized in that the mixture of analyte and α-cyano-4-hydroxy cinnamic acid is centrifuged and dried at room temperature.
The method for quantitative analysis of 17α-hydroxyprogesterone according to claim 1, wherein the internal standard 17α-hydroxyprogesterone- d ₈ is added at a concentration of 20 to 200 ng / mL in serum.

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